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. 2019 Mar 29;9:5371. doi: 10.1038/s41598-019-41853-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Knockdown of GSK-3β attenuated mitochondrial ROS production and cell death of the GAS-infected RAW264.7 cells. The expression of GSK-3β was silenced in RAW264.7 cells using lentiviral-based shRNA (shGSK-3β) construct and the luciferase shRNA construct was using as negative control. The transfected cells and RAW264.7 cells (without transfection) were infected with GAS at a MOI of 25 as described in Materials and Methods. (a) Mitochondrial ROS levels were measured by MitoSOX staining at 3 h post GAS infection. Fluorescence intensity % was shown and expressed as described in Materials and Methods. Results are represented as mean ± SD. In GAS-infected groups, ***P < 0.001 compared with shGSK-3β knockdown group versus negative control group (One-way ANOVA test followed by Tukey’s test; n = 4) or versus the group of RAW264.7 cells (One-way ANOVA test followed by Tukey’s test; n = 4). (b) The GAS-mediated cell deaths were measured by LDH release at 18 h post GAS infection, as described in Materials and Methods. **P < 0.01 compared with shGSK-3β knockdown group versus negative control group (One-way ANOVA test followed by Tukey’s test; n = 4) or versus the group of RAW264.7 cells (One-way ANOVA test followed by Tukey’s test; n = 4).